The present invention relates to an array of individually addressable micro-electrodes for performing biological, synthetic and diagnostic reactions. Specifically, the present invention can be used for diagnostic assays such as nucleic acid hybridizations or antibody-antigen reactions and for reactions using synthetic molecules such as the preparation of carbohydrate polymers, as well as for other multimolecular reactions.
The detection and monitoring of biomacromolecules, such as nucleic acid chains and proteins, is a growing sector of the clinical diagnostic field which assays human physiological fluids. In particular, recent concern has centered on the necessity of producing large numbers of new assays for specific nucleic acid sequences as the entirety of the human genome becomes sequenced. Furthermore, because the screening of unknown pathogens or elicited immunoglobulins requires assaying a large number of synthetic or natural epitopes, physiological fluid samples are preferably simultaneously reacted with large numbers of epitopes in order to more efficiently diagnose a particular disease or condition. These needs are not currently met by conventional diagnostic techniques.
For example, conventional molecular biological techniques such as Northern blots and DNA sequencing, and immunological techniques such as ELISA, presently require laborious handling procedures by qualified technicians which is often time-consuming and lacking in sensitivity, specificity or reproducibility. Furthermore, relatively few conventional methods of assaying DNA lend themselves to the massively parallel assays which are needed to assay many samples simultaneously.
Such assays have been attempted with nucleic acid hybridization, in which a target nucleic acid sequence is attached to a support, and is then reacted with a complementary probe sequence. Typically, the probe carries a reporter group such as a fluorophore or radioactive label, which is then detected in order to determine if the probe has bound to the target. The equivalent assay for proteins is the microtiter ELISA immunoassay. However, both nucleic acid hybridizations and ELISA immunoassays have a number of drawbacks as currently performed.
First, a relatively high concentration of probe-target complexes must be achieved in order to accurately detect the reporter groups. This in turn requires large amounts of reagents which may be expensive, scarce or both. Second, even relatively miniaturized assays, such as a microtiter ELISA immunoassay, require rather large surface areas for each individual reaction. Thus, performing a massively parallel assay can result in an unacceptably large overall surface area for the entire assay as a whole, which also tends to increase the amount of required reagents.
In order to overcome these problems, recently attempts have been made to miniaturize these assays to the micron level, thereby producing a "chip" which could hold all of the samples needed for one assay in a microscopically small area. Research has been particularly active in the field of nucleic acid hybridization. For example, a number of methods have been described for binding target DNA to a support surface at high densities, and even for synthesizing target DNA or peptide segments directly on a support surface. These methods all enable relatively high density arrays of target material to be prepared for hybridization by complementary DNA or peptidic probes.
In one method, starter oligonucleotides were attached to glass slides [E. M. Southern, Nuc. Acids Res., 22:1368-1373, 1994]. In subsequent synthetic steps, these oligonucleotides were elongated by applying reagents in a defined area. After the synthesis is complete, complementary probes were hybridized to the target DNA on the slide. Similarly, arrays of target DNA were synthesized on aminated polypropylene film using a controlled photodeprotection chemistry and photoprotected N-acyl-deoxynucleoside phosphoramidites [R. Matson, Anal. Biochem., 224:110-116, 1995]. Arrays of peptides have been synthesized on cellulose sheets [R. Frank, Tetrahedron, 48:9217-9232, 1992]. Methods which do not include direct synthesis on the support have also been developed, which involve the attachment of PCR products to silylated glass slides [M. Schena, PNAS, 93:10614-10619, 1996]. Thus, attempts have been made to increase the density of target DNA or peptides by miniaturizing the size of individual sites in the array.
Unfortunately, none of these methods results in a target material-support complex which is easy to hybridize with a complementary probe. Although the DNA or peptide fragments may be in a high density array, the support itself must still be laboriously handled--washed, incubated with complementary probes and then analyzed with a detection device which can determine if the probe bound to the target. Such miniaturization still does not remove many of the obstacles to easy and rapid examination of samples, including the need to perform many subsequent labor intensive steps. Thus, mere miniaturization through high density arrays is not a complete solution to current analytical needs.
A further step towards obtaining such a solution is the APEX (automated programmable electronic matrix) chip as disclosed in U.S. Pat. No. 5,605,662. APEX is a silicon chip containing an array of many micro-electrodes, each of which has a different DNA segment attached. Electric currents are used to both increase the specificity and efficiency of preparation of the chip, and to increase the specificity and rapidity of the DNA hybridization reaction. The chip can be used as part of a highly automated method of performing DNA hybridization reactions. Unfortunately, the APEX technology is still lacking in a number of aspects.
First, obtaining accurate results with this chip requires a high degree of specificity for the initial attachment of target DNA segments onto the micro-electrodes. Since these micro-electrodes are very small and the resultant array is very dense, such specificity cannot be guaranteed by spatial separation of solutions containing the different target segments. Instead, electronic activation of different micro-electrodes is used to attach specific target DNA segments to different micro-electrodes. However, the remaining electrodes are still available to the target DNA segments during this process. Thus, there is no absolute bar to prevent DNA segments from attaching to more than one electrode simultaneously.
Second, the orientation of the attached DNA segments is not considered. Since the absolute amount of target DNA at each electrode is relatively low, the chip depends upon a relatively high rate of probe/target interactions to enable sufficient signals for determining whether a particular probe bound to a particular target. In any type of reaction which binds target DNA to a support, some of the bound DNA will not be accessible to a complementary probe because of the relative orientation of the target DNA to the probe. Ensuring the proper orientation of the target DNA segments will reduce the amount of inaccessible target DNA. However, U.S. Pat. No. 5,605,662 does not discuss this issue and certainly does not provide any solutions to this important problem. Thus, the efficacy of the technology disclosed in U.S. Pat. No. 5,605,662 could be potentially limited by is the problem of orientation of the target DNA segments.
Finally, U.S. Pat. No. 5,605,662 does not describe a method for detecting the presence of a complementary probe bound to a target DNA segment in any detail. Clearly, the overall miniaturization of the array of target DNA segments, coupled with the relatively low amount of probe molecules which could bind to any one target, makes the issue of detection of binding highly important. Thus, the lack of discussion and description of this issue is a significant problem.
Therefore, there is an unmet need, and it would be highly useful to have, a device for performing hybridizations and other multimolecular reactions and interactions, which employs a high density array of micro-electrodes, which specifically and efficiently binds target material such as DNA segments in a proper orientation, which can easily and rapidly be used for subsequent molecular interactions such as hybridization of complementary probes, and which is part of a system for rapid and sensitive detection of such molecular interactions.